Simultaneous spectrofluorimetic determination of remdesivir and simeprevir in human plasma

As new infectious mutations of SARS-CoV-2 emerged throughout the world, innovative therapies to counter the virus-altered drug sensitivities were urgently needed. Several antiviral options have been in clinical trials or in compassionate use for the treatment of SARS-CoV-2 infections in an attempt to minimize both clinical severity and viral shedding. Recent research indicated that simeprevir acts synergistically with remdesivir, allowing for a multiple-fold decrease in its effective dose when used at physiologically acceptable concentrations. The goal of this work is to develop a sensitive synchronous spectrofluorimetric approach to simultaneously quantify the two drugs in biological fluids. Using this method, remdesivir and simeprevir could be measured spectrofluorimetrically at 283 and 341 nm, respectively, without interference from each other using Δλ of 90 nm. The effect of various experimental parameters on the fluorescence intensity of the two drugs was extensively explored and optimized. For each of remdesivir and simeprevir, the method exhibited a linearity range of 0.10–1.10 μg/mL, with lower detection limits of 0.01 and 0.02 μg/mL and quantification limits of 0.03 and 0.05 μg/mL, respectively. The high sensitivity of the developed method permitted the simultaneous determination of both drugs in spiked plasma samples with % recoveries ranging from 95.0 to 103.25 with acceptable standard deviation values of 1.92 and 3.04 for remdesivir and simeprevir, respectively. The validation of the approach was approved by the International Council of Harmonization (ICH) guidelines.

www.nature.com/scientificreports/ viral NS3/4A protease, which prevents viral maturation by interfering with protein synthesis 20 . In-vitro, SIM decreases SARS-CoV-2 viral load and inhibits not only the main protease (M pro) but also the RNA-dependent RNA polymerase (RdRp), as well as modulating host immunological responses 19,21 . Interestingly, SIM and REM work synergistically, allowing the effective dose of both to be reduced 19 . The unexpected anti-SARS-CoV-2 mechanism of SIM might contribute to future antiviral strategies. Also, this combination therapy may also assist in reducing the quantity of medication needed to alleviate harmful effects 19 . For the analysis of SIM, the literature review reveals several analytical methods for its assay, whether alone or in combination. These methods involve spectrophotometric 22,23 , chemometric-assisted spectrophotometric 24 , spectrofluorimetric [25][26][27] , and LC methods [28][29][30][31][32][33] . Currently, no method has been reported for simultaneously analyzing REM and SIM in biological fluids as co-administered medications. This means that hospitalized in-patients need a way to measure both medications at the same time so that their therapeutic drug monitoring can be evaluated.
When scanning each REM and SIM on the classical spectrofluorimetric approach, it was noticed that their two emission spectra were intensively overlaid preventing this approach from being used for resolution. Using a constant wavelength synchronous spectrofluorimetric (SFS) method, the problem of spectral overlap was handled.
Although spectrofluorimetric technology is known for its great sensing properties, selectivity issues might occur when studying multi-component combinations due to the overlay of broadband spectra. This overlaying difficulty might be confronted by the employing synchronous technique, as it has several of benefits over standard fluorescence techniques, which include high selectivity, proper spectra, and minimum intrusion [34][35][36] . SFS is highly basic but proved to be an effective approach for the quantitative assessment of the two drugs in an appropriate time frame because of its narrow and sharp spectra [37][38][39][40] . REM and SIM emit separable synchronous  www.nature.com/scientificreports/ fluorescence emission peaks with intensities measurable at 283 nm and 341 nm, respectively, after adopting the constant wavelength synchronous spectrofluorimetric (Δλ) method. Centered on the pharmacological and chemical characteristics of the aforementioned medications and their enhanced therapeutic efficacy in the management of COVID-19 illness and for the goal of therapeutic medication monitoring, the SFS methodology was exploited for the concomitant quantification of REM and SIM in spiked plasma samples, and the results were satisfactory.

Experimental
Instruments and material. Instrumentation.
-An Agilent G8900A Cary Eclipse Spectrofluorimeter fitted with a Xenon flash lamp was used for the study (Agilent, California, USA). The synchronous method was set using Δ λ = 90 nm, slit width of 5 nm, and factor 20 was utilized to smooth out the obtained spectra. The voltage was 800 V, and the scanning rate was 600 nm/ min in the wavelength range of 200-600 nm. -Microsoft Excel (2010) was used for creating figures and graphs from the data stored in the ASCII. format. -The pH meter from Consort NV P-901 Belgium was used to adjust the pH of the buffer solutions used.
-The biological samples were mixed with a Vortex IVM-300p from Gemmy Industrial Corporation in Taiwan, then separated with a centrifugal force from Germany's version 2-16P.
-The raw material of remdesivir was generously donated by EVA Pharma. Co., Cairo, Egypt.
-The raw material of simeprevir sodium was kindly given by AUG Pharma (2nd Industrial Zone, 6 October City, Egypt). -HPLC grade organic solvents were obtained from a Fisher Scientific distributor in Cairo, Egypt.
-Different surfactants were purchased from Sigma-Aldrich, Germany.
-El-Nasr Pharmaceutical Chemicals Co., Cairo, Egypt, supplied all other chemicals: acetic acid (96%), phosphoric acid, boric acid, and sodium hydroxide. -The plasma sample was collected from Mansoura University Hospital's National Egyptian Blood Bank, Mansoura, Egypt. After mild thawing, it was kept frozen at -20 °C until required. Procedures. Construction of calibration graphs. Aliquots of REM and SIM standard solutions within the working concentration range listed in Table 1 were transferred into a set of 10 mL volumetric flasks, diluted to volume with ethanol, and mixed well. The synchronous fluorescence spectra of the prepared solutions were measured at a wavelength difference of 90 nm. The fluorescence intensities were recorded at 283 nm and 341 nm for REM and SIM, respectively. At the same time, a blank solution was carried out. The calibration plots were then created by plotting relative synchronous fluorescence intensity (RSFI) versus ultimate drug concentrations, and then the relevant regression equations were derived.
Analytical procedure for REM/SIM in lab-prepared mixtures. To verify the method's specificity, standard solutions encompassing multiple proportions of REM and SIM were diluted in 10 mL volumetric flasks with ethanol to reach the appropriate concentration. The nominal content was determined using its corresponding regression equation. www.nature.com/scientificreports/ assessment was carried out as mentioned under "Construction of calibration graphs". Using the appropriate regression analysis, the nominal plasma content was estimated.
Ethical approval. No human subjects or experimental animals were involved in this study. No real plasma samples were included in this study. Plasma sample was "pooled plasma", obtained from the National Egyptian Blood Bank at Mansoura University Hospital, Mansoura, Egypt.

Results and discussion
Based on the relevance of SFS in pharmaceutical analysis, a sensitive and simple spectrofluorimetric approach for measuring REM and SIM in spiked plasma samples was created. As shown in Fig. 2, both ethanolic solutions of REM and SIM emit fluorescence at wavelengths of 400 and 429 nm upon excitation at 243 and 284 nm, respectively. Detailed examination of Fig. 2 reveals that the REM and SIM emission spectra are significantly overlapped, rendering simultaneous analysis of such a combination a challenging task. As a result, it was recommended to use the extremely sensitive and selective SFS approach to enhance its selectivity. Typical SF spectra for both REM and SIM were reported at Δλ of 90 nm, and it was found that their peaks were well-defined, allowing each drug to be analyzed without interference from the other. The synchronized fluorescence intensity of REM and SIM could be measured with high sensitivity at 283 nm and 341 nm, respectively (Fig. 3). Different concentrations of REM were measured at 283 nm in the presence of a fixed concentration of SIM (0.60 μg/mL) as shown in Fig. 4, whereas SIM was detected at 341 nm in the presence of a fixed concentration of REM (0.40 μg/mL) as shown in Fig. 5. All of the fluorescence measurements were obtained at Δλ of 90 nm.
Optimization of experimental factors. The effect of various investigational conditions on the fluorescence intensities of the medications described above was explored and optimized. Each variable was evaluated independently, while the others remained unchanged. The value of Δλ was found to deeply affect the SF scanning process. It also has a substantial impact on sensitivity as well as peak resolution. As shown in Fig. S1, both   www.nature.com/scientificreports/ REM and SIM were scanned at various Δλ values ranging from 20 to 160 nm. The SF spectra of the indicated drugs were produced in a single step with reasonable sensitivity using Δλ value of 90 nm, which proved to be appropriate as it gave a clear response for both substances. Fluorescence intensities for both medications were reduced when Δλ was lowered below 90 nm. At Δλ above 90 nm, both medications fluoresced intensely but with overlapped spectra. The optimal conditions for fluorescence signal and segregation of the two overlapped spectra were determined using a diversity of solvent solutions, including distilled water, acetone, acetonitrile, methanol, and ethanol. Both drugs were scanned at Δλ = 90 nm in different solvents and compared in term of sensitivity as well as spectral resolution. When it came to REM, ethanol had the greatest fluorescence values, followed by acetonitrile and methanol, respectively, while water had the lowest fluorescence signal of all the diluents tested. When it comes to SIM, distilled water generated relatively low fluorescence readings related to the organic solvents, including; ethanol, acetonitrile, and methanol. Acetone also significantly reduced the SFIof both drugs. Because of this, ethanol was adopted in this study as the principal diluent owing to sensitivity and environmental concerns, as shown in Fig. S2. Britton Robinson buffer solutions with a pH range of 2.1-12.0 were employed in this investigation. It was noticed that changes in pH had no effect on the SFI of both drugs over the whole pH range. This result matches with literature 9 in which REM was determined utilizing its native fluorescence at 244/405 nm. It was reported that REM fluorescence intensity was studied using the BRb buffer and exhibited nearly similar intensities within the pH range of 3.0-5.0 and such intensity started to decrease above pH 6.0. As a result, no buffer solutions were used in the suggested method. Different organized media were studied, using non-ionic surfactant (Tween-80), anionic surfactant (SDS), anionic polysaccharide (CMC),  www.nature.com/scientificreports/ macromolecules (β-CD) and cationic surfactant (cetrimide). All are prepared in distilled water at concentrations greater than the critical micelle level and tested to see if they can improve fluorescence readings of the aforementioned drugs 41 . As shown in Fig. S3, neither surfactants nor macromolecules resulted in a considerable increase in the fluorescence emission of REM when compared to ethanol as the selected green diluent. This observation may be explained by the bulky nature of the drug and consequently the steric hindrance preventing its inclusion in the cavities of the micelles. While for SIM, only tween-80 produces a significant increase in fluorescence signal compared to other surfactants and macromolecule. This signal is nearly equal to that of ethanol. As a result, in the proposed method, ethanol was chosen as the best solvent for both drugs without the use of a buffer or organized media.

Method validation.
To determine whether the proposed approach was valid or not, the International Conference on Harmonization (ICH) Q2 (R1) guidelines were adopted 42 .
Range and linearity. For both medications, a linear correlation was established between the values of relative synchronous fluorescence intensity (RSFI) and the ultimate drug concentrations across the range of 0.10-1.10 μg/mL. Table 1 presents a summary of the obtained ranges, regression equations, and calibration curve data. The calibration curves' linearity was confirmed by statistical analysis of the data 43 and values approaching the unity of correlation coefficients. Table 1. The acquired values demonstrated that the suggested approach can accurately quantify the previously stated medications in spiked plasma and, hence, may be used in future in vivo research.

Limit of quantitation (LOQ) and limit of detection (LOD). LOQ and LOD values are depicted in
Accuracy and precision. The suggested method's accuracy was confirmed by statistically analyzing the obtained data compared to those obtained using reference methods 9,25 . The Student t-test and the Variance ratio F-test were used to statistically examine the data 43 . The results in Table 2 reveal that there was no substantial difference in performance between them in terms of precision and accuracy. The precision of the developed approach was evaluated to determine inter-day and intra-day precisions. The medications under study had low percent RSD and percent error, indicating that the established procedures are sufficiently precise (Table 3).
Selectivity. The anticipated methodology was utilized to analyze REM and SIM at the same time with good recoveries and without interference, as cited in Table 4. The recommended approach's selectivity was further proven by quantifying the cited drugs in complicated human plasma matrices. The procedure was demonstrated to have adequate selectivity to examine the previously stated drugs with high percent recoveries (95.0-103.25%) and low percent RSD values, pointing out that there was no disruption from plasma intrinsic constituents.
Applications of the suggested procedure. REM/SIM analysis in lab-prepared mixtures. As shown in Fig. 6, the indicated process was utilized to analyze synthetic combinations with varied REM and SIM proportions. The amounts of the specified medications in their synthetic mixtures might be calculated using the corresponding regression equations. The obtained results, as shown in Table 4, revealed the accuracy of the designated procedure.

Analysis of human plasma samples spiked with REM and SIM.
For the treatment of Covid-19, Gilead recommends a 200 mg loading intravenous dose of REM on the first day followed by nine days of 100 mg once daily to target viral exposures in plasma and cells 44 . It was documented that C max of REM on the first day was 5.44 μg/mL and on the fifth day, it was 2.61 μg/mL 44 . While C max of SIM was reported to be approximately 2.588 μg/mL after multiple 150 mg once-daily oral doses, with a t max of 4-9 h 45 . The high sensitivity of the developed techniques  Table 3. Inter-day and intra-day precision data for the studied drugs using the proposed method. www.nature.com/scientificreports/ SIM, respectively, implying that it might be used for in-patient therapeutic drug monitoring of the specified drugs as illustrated in Fig. 7.

Concentration (μg/mL)
Estimation of the suggested method's greenness. Globally, there is an increased emphasis on managing waste and dangers to enhance green analysis. Analytical GREENNESS metric approach (AGREE) 46 and Green Assessment Profile Index (GAPI) 47 are two green metrics that were used in this study. Regarding AGREE, it is a technique for identifying the environmental and occupational dangers involved in the analytical procedure. It provides a clock-shaped chart with a circumference split into 12 pieces, based on the 12 principles of Green Analytical Chemistry 48 . On a color scale, each item is handled as a separate parameter. The center of the AGREE chart indicates the overall acceptance color and assessment score on a scale of 0 to 1 (Fig. 8). A semiquantitative technique called GAPI was also employed for both drugs in raw materials and spiked plasma samples in order to ascertain the green property in each phase. It employs a visual graph to categorize the greenness of each phase in a methodological approach, using a chromaticity scale of red, yellow, and green. The two processes used generate little waste and require only a small amount of non-toxic chemicals. Additionally, the applied procedure was a direct approach that was intended for qualification and quantification. The results, which are adequate and indicate acceptable green approaches, are displayed in Fig. 8.

Comparison between the proposed method and other reported methods. The proposed method
involved the simultaneous determination of a synergistic combination of REM and SIM. The resolution of the overlaid spectra was allowed by using Δλ value of 90 nm, which is not equal to the difference between the emission and excitation wavelengths of the two drugs. Therefore, the sensitivity of the proposed method is slightly lower than the native estimation reported in the literature 9,25 . However, it is higher than another fluorimetric method reported for SIM 25 . As a result, the key advantage of the new spectrofluorimetric technique is energy savings, as the spectrofluorometer requires the least amount of energy (less than 0.1kWh) when compared to HPLC (approximately 1.5kWh) in addition to high sensitivity of the proposed method compared to reported HPLC 12,13,32,33 . Despite the fact that the chromatographic technique is widely employed in pharmaceutical qual-  www.nature.com/scientificreports/ ity control and research laboratories, it is still considered a sophisticated technology due to the high cost of the equipment (column and detector) and the environmental damage caused by the use of hazardous organic solvents. Also, the spectrophotometric techniques are widely used in routine work analysis because of their speed, ease of use, and relative inexpensiveness, but they are deficient in terms of sensitivity 8,23,24 . Additionally, the proposed method is significantly easier, greener (as verified by AGREE and GAPI), cheaper, and time-saving, as proven in (Table 6).  www.nature.com/scientificreports/

Conclusion
The determination of the co-administered antiviral drugs in biological fluids represents a very imperative and pertinent step in the treatment of SARS-CoV-2 illness, and this requires therapeutic drug monitoring. This work analyzes remdesivir and simeprevir concurrently without interference from plasma endogenous components using a very green-sensitive synchronous spectrofluorimetric method. The utilized instrument is straightforward and inexpensive compared to other tools, such as HPLC and gas chromatography. The solvent used in the suggested technique is ethanol, which is green, less expensive, and more widely accessible. Because of the environmental benefits of using green solvents, the technique can be regarded as a viable quality control alternative.

Data availability
All the data generated or analysed during this study are included in the Supplementary File.